ADP-ribosylation factor domain protein (ARD1) initially cloned in this laboratory, differs from other ARFs having a 46-kDa N-terminal extension (p5), which functions as a GTPase-activating protein (GAP) for the C-terminal ARF domain (p3). Like ARF GAP proteins, the GAP domain of ARD1 contains a zinc-finger motif and arginine residues that are critical for activity. It differs from other ARF GAPs in its covalent association with the GTP-binding domain and the specificity of its GAP activity for the ARF domain of ARD1. Study of a putative alternatively spliced mRNA and protein product is being completed. ARFs are presumed to play a key role in the formation of intracellular transport vesicles and their movement from one compartment to another. Both overexpressed and endogenous ARD1 were associated with Golgi and lysosomal membranes, consistent with a role in protein trafficking among these compartments. ARD1 appears to be activated specifically by cytohesin-1, and residues responsible for specificity of the cytohesin-1/ARD1 interaction were identified. Characterization of multiple phenotypes in ARD1 "knock-out" mice, which had been slowed by problems with reproduction after repeated back-crossing, is again proceeding.